The immune system has evolved to defend the host from invading microorganisms. It achieves this via both innate and adaptive responses. In both cases, leucocyte migration to sites of infection, and from sites of infection to secondary lymphoid organs is critical for an adequate immune response. In addition, interactions between leucocytes, for example an antigen presenting cell and a CD4 T cell, are also essential for their activation and optimal function. Actin and membrane remodelling are required for many of these important dynamic processes. Breast cancer-associated protein 1 (BRAP-1) (also known as Bridging integrator-2 (Bin2)) is an N-BAR domain containing protein that is highly expressed in leucocytes. This family of proteins play an important role in membrane curvature and remodelling1.

We recently showed that BRAP-1 is associated with actin rich structures on the plasma membrane, including podosomes and the leading edge of migrating cells, and is enriched at the immune synapse in B cells (figure 1, 2). SiRNA knockdown of endogenous protein led to decreased cell migration, increased phagocytosis and reduced podosome density in myeloid cell lines. In contrast, overexpression of BRAP-1 led to decreased phagocytosis and increased podosome density and dynamics2.

Further studies will be required to understand the role of this protein in determining the dynamic behaviour of leucocytes in vivo under both physiological and pathological conditions.

Proposed project:

A BRAP-1 knockout mouse is currently being generated (in conjunction with the Sanger International Knockout Mouse Consortium). The student will characterise the phenotype of this novel knockout mouse, and use it to answer a number of specific questions about the role of BRAP-1 in leucocyte migration and activation in vivo. Studies will focus on:

1. Basic immunophenotyping of the BRAP-1 knockout (characterisation of primary and secondary lymphoid organs by flow cytometry and immunohistochemistry and confocal microscopy).

3. Investigation of the effect of BRAP-1 deficiency on B cell interaction with CD4 T cells during an immune response. 2 photon, in vivo imaging will be used to visualise lymph nodes following transfer of fluorescently labelled cells.

4. Investigation of the role of BRAP-1 in defence against bacterial infection, in vivo. Mice will be infected with Strep pneumoniae and responses determined. In vivo, 2 photon imaging of neutrophil migration in response to dermal TLR-ligand and bacterial innoculation will be performed. Further mechanistic studies, including in vitro phagocytosis assays will also be required.

Hypoxia augments allergic inflammation and contributes to steroid-resistance in asthma.

Background and Preliminary Data

Glucocorticoids are the mainstay of asthma pharmacotherapy, but severe asthma is associated with steroid-resistance; such steroid-resistant asthmatics account for a disproportionate utilisation of health care resources (up to 50% of the total costs for asthma1). Systemic hypoxia occurs during acute asthma exacerbations; more importantly, local tissue hypoxia is a prominent feature of inflammation and has been demonstrated in the context of allergen challenge in human asthmatics2. We have shown that hypoxia modulates multiple granulocyte functions3-5, renders eosinophils (the key cellular effectors of allergic inflammation6) resistant to glucocorticoid-mediated apoptosis, and upregulates their release of pro-inflammatory cytokines such as IL-8 (unpublished observations); this may lead to prolonged eosinophil survival in allergic inflammation and compound the accompanying neutrophilic inflammation.

Project Outline

The project will focus on the following key objectives:

To establish the composition of the hypoxic eosinophil secretome (in the presence and absence of glucocorticoids) using a proteomics-based approach, with the aim of identifying biologically relevant and potentially ‘druggable’ targets.

To investigate the biological activity of supernatants derived from hypoxic eosinophils on other relevant inflammatory cells including neutrophils, macrophages and T-cells.

To explore the mechanistic basis of hypoxia-induced glucocorticoid resistance by analysis of the hypoxic and normoxic eosinophil transcriptome and kinome, and by the targeted use of relevant inhibitors.

To study (in collaboration with Professor Nicholas Morrell and Professor Randall Johnson) the effects of hypoxia on airway hyperreactivity and airway inflammation in response to ovalbumin challenge (a well-established mouse model of asthma) in steroid-naive and steroid-treated animals, using HypoxyprobeTM staining to quantify tissue hypoxia.

Exploring the mechanisms that underpin glucocorticoid resistance may lead to novel treatments for this challenging clinical problem.

Division: Respiratory Medicine Mutations in the bone morphogenetic protein type 2 receptor (BMPR2) are the commonest genetic cause of pulmonary arterial hypertension, a devastating disease that affects young people leading to death within a few years from diagnosis. Our group have determined that BMPR2 forms a receptor complex on endothelial cells with the type I receptor, ALK1 and that the ligand for this receptor complex is bone morphogenetic protein 9 (BMP9). In knockin mouse models of BMPR2 mutation we have shown that BMP9 administration can reverse established disease. This project will further determine the role of BMP9 in PAH. Specifically, we will determine the mechanisms regulating BMP9 expression in the liver (the source of circulating BMP9), since pulmonary hypertension is associated with reduced BMP9 transcription in the liver and determine the therapeutic benefit of adenoviral gene therapy to overexpress BMP9 in the liver in mouse and rat models of PAH. We have further determined that biological activity of BMP9 is regulated by neutrophil elastase within the lungs. This project will determine the contribution of altered BMP9 production and degradation in mouse models, to revel novel interventions to treat patients with PAH.

Principal Investigator:Dr Sergey Nejentsev

Division of Infectious Diseases

Genetic and functional mechanisms of Primary Immunodeficiencies

Summary

Primary Immunodeficiencies are a heterogeneous group of more than 200 diseases caused by Mendelian mutations in the immune genes. Primary Immunodeficiencies manifest as severe recurrent infection and often can be life-threatening. Mutations in some patients have been identified previously using linkage analysis and candidate gene approach. These discoveries proved to be extremely informative for understanding of the human immune system. Nevertheless, genetic causes in the majority of patients with Primary Immunodeficiencies remain unknown.

The aims of the proposed PhD project are to discover new causative mutations in patients with Primary Immunodeficiencies, to investigate functions of the affected proteins and to uncover new mechanisms of susceptibility to infection. Initially, advanced methods of human genetics will be employed, such as sequencing of exomes and whole genomes of patients, focusing primarily on those suffering from mycobacterial infections. Then a variety of molecular and cell biology techniques will be used, including quantitative PCR, Western blotting, cytokine secretion analyses, immunofluorescence confocal microscopy, electron microscopy, RNA interference, gene cloning and in vitro infection models. These techniques are established in the laboratory and training will be provided. Discovery of new genetic and functional mechanisms of Primary Immunodeficiencies will improve understanding of the immune system and will allow to design new diagnostic assays and new therapeutic approaches that will save patients’ lives.

Division of Infectious Diseases, School of Clinical Medicine, University of Cambridge

A major cause of disease in a number of immune-compromised populations, the molecular mechanisms that control human cytomegalovirus (HCMV) latency and reactivation are still not fully understood. Currently, the working model for HCMV is that latency is established in a pluripotent pool of haematopoietic cells resident in the bone marrow. Despite the pluripotency of the progenitor, HCMV carriage is observed predominantly in the cells of the myeloid lineage with reactivation occurring upon terminal differentiation of these cells which, in healthy individuals, is effectively controlled by a robust immune response.

HCMV latency is defined by an absence of lytic gene expression and infectious virus release. However, it is becoming increasingly clear from our work and others that a subset of viral genes are expressed during latency which elicit a highly polarised immune-modulatory T cell response. The focus of this project will be on LUNA, a viral protein expressed during HCMV latency, with an emphasis on understanding the protein function at a molecular level and how the immune response to this protein during latency is regulated. Specifically, we have identified a de-sumoylase function associated with LUNA which promotes the dispersal of PML bodies in latently infected cells which impacts on HCMV reactivation via regulation of the major immediate early promoter (MIEP) in a chromatin-dependent manner. Consequently, this project would have 3 major aims. i) Further characterisation of the de-sumoylase function with particular emphasis on novel cellular targets we have recently identified ii) To understand the regulatory role LUNA has on latent viral gene expression which, like the MIE genes, are also regulated by higher order chromatin structure and finally, iii) the characterisation of the immune response to latently expressed antigens and how this could be manipulated to provide a paradigm for immune-mediated clearance of HCMV.

Overall this project would expose the student to a wide range of techniques providing excellent training in the areas of virology, molecular biology, cell signalling and immunology and is aligned with the broader aims of the ongoing research in the Infectious Diseases group and will be performed in collaboration with the groups of Dr Mark Wills and Professor John Sinclair..